Apparatus for screening compound libraries

ABSTRACT

Disclosed are apparatus for screening compound libraries using frontal chromatography in combination with mass spectrometry to identify and rank those members of the library that bind to a target receptor. The apparatus of this invention also permit a compound library to be rapidly screened to determine if any member of the library has an affinity for the target receptor as measured by a pre-selected indicator compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/069,890,filed Apr. 29, 1998, now U.S. Pat. No. 6,054,047, issued Apr. 25, 2000,which application claims the benefit of U.S. Provisional Application No.60/079,622, filed Mar. 27, 1998. Each of these applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to apparatus for screening compound libraries,such as compound libraries generated using combinatorial chemistrytechniques. The apparatus of this invention employ frontalchromatography in combination with mass spectrometry to screen a libraryof compounds to identify and rank those members of the library that bindto a target receptor. The apparatus of this invention also permit acompound library to be rapidly screened to determine if one or moremembers of the library have an affinity for a target receptor asmeasured by a pre-selected indicator compound.

2. References

The following publications, patents and patent applications are cited inthis application as superscript numbers:

¹K. S. Lam, Anti-Cancer Drug Des. 1997, 12, 145-167.

²P. M. Sweetnam et al., In Burger's Medicinal Chemistry and DrugDiscovery; M. E. Wolff, Ed.; John Wiley & Sons: New York, 1995; pp697-731.

³R. H. Griffey et al., In Proceedings of the 45^(th) ASMS Conference onMass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5,1997; p. 400.

⁴L. Fang et al., In Proceedings of the 45^(th) ASMS Conference on MassSpectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5, 1997; p.401.

⁵Y.-H. Chu et al., J. Am. Chem. Soc. 1996, 118, 7827-7835.

⁶Y.-Z. Zhao et al., J. Med. Chem. 1997, 40, 4006-4012.

⁷Y. F. Hsieh et al., J. Mol. Div. 1996, 2, 189-196.

⁸R. W. Nelson et al., Anal. Chem. 1995, 67, 1153-1158.

⁹D. C. Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387.

¹⁰PCT/US97/07964 (International Publication No. WO 97/43641), publishedNov. 20, 1997, entitled “Molecular Diversity Screening Device andMethod.”

¹¹R. Wieboldt et al., Anal. Chem. 1997, 69, 1683-1691.

¹²R. B. van Breemen et al., Anal. Chem. 1997, 69, 2159-2164.

¹³ M. L. Nedved et al., Anal. Chem. 1996, 68, 4228-4236.

¹⁴PCT/US95/03355 (International Publication No. WO 95/25737), publishedSep. 28, 1995, entitled “Method for Identifying Members of CombinatorialLibraries.”

¹⁵PCT/EP97/02215 (International Publication No. WO 97/43301), publishedNov. 20, 1997, entitled “Identification of Members of CombinatorialLibraries By Mass Spectrometry.”

All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

State of the Art

In recent years, a large number of combinatorial chemistry techniqueshave been developed which permit vast libraries of diverse chemicalcompounds to be rapidly synthesized.¹ In combinatorial chemistry, aseries of chemical reactions is typically conducted employing aplurality of reagents at each step to generate a library of compounds.Such techniques have the potential to greatly accelerate the discoveryof new compounds having biologically useful properties by providinglarge collections of diverse chemical compounds for biologicalscreening.

This ability to rapidly generate large collections of compounds usingcombinatorial chemistry techniques has created a need for new methods ofscreening compound libraries. The traditional approach of screening eachcompound individually in an assay to identify those compounds having thedesired biological activity is no longer practical due to time andresource constraints. Thus, a need exists for new methods and apparatuswhich permit the rapid screening of compound libraries.

In this regard, various methods for screening compound libraries havebeen reported. Typically, these screening methods involve the use oftarget receptors which have been labeled with fluorescent or otherreporter groups.² In these methods, the compound library, typicallybound to a resin bead, is exposed to the labeled target receptor andthose members binding to the labeled target receptor are identified andphysically separated. The particular ligand binding to the targetreceptor is then identified. In many of these techniques, elaborateprocedures are required to keep track of individual members of thelibrary. For example, coded tags are often added during the synthesis ofthe combinatorial library to allow the structure of the individualmembers to be subsequently determined. Alternatively, combinatoriallibraries can be prepared in an array and the individual members of thelibrary subsequently identified by their location in the array. Whilesuch methods can be effective, the need to keep track of individualmembers of the library during their synthesis and screening is quitecumbersome and often limits the type of synthetic procedures that can beemployed. Additionally, many of these techniques require that thesynthetic procedures be conducted on a solid phase, thus furtherlimiting the synthetic procedures and reagents that can be used.

As an alternative, mass spectrometry is emerging as an important toolfor the interrogation of combinatorial libraries. To date, massspectrometry has been used to assess library quality^(3,4) and, whencoupled with molecular recognition technologies, has allowed for somesuccess in the isolation and characterization of active librarycompounds.⁵⁻¹⁵ Typically, when screening compound libraries forbiologically active members, mass spectrometry is used in combinationwith a “capture and release” methodology. In this methodology, compoundmixtures are presented to the target receptor, which is oftenimmobilized on a solid support, and the resulting ligand-receptorcomplexes are separated from the unbound members of the library. Afterseparation, the ligand-receptor complexes are typically denatured, forexample, with a solvent and the solvent mixture containing thepreviously bound ligands is presented to the mass spectrometer to permitidentification of the high affinity ligands.

For example, ultrafiltration has been used in combination withelectrospray mass spectrometry to screen combinatorial libraries.¹⁰⁻¹²In this method, ligands present in a compound library are allowed tobind to a receptor and the resulting ligand-receptor complexes arepurified by ultrafiltration. The ligand-receptor complexes are thendissociated using a solvent, such as methanol, and the previously boundligands are detected by an electrospray mass spectrometer.

Affinity capillary electrophoresis (ACE) has also been coupled with massspectrometry to screen combinatorial libraries.⁵ In this procedure, ACEis used to separate ligand-receptor complexes from unbound ligands andmass spectrometry is used to identify the high affinity ligands.

Similarly, compound libraries have been screened using affinitychromatography in combination with mass spectrometry. For example, WO97/43301 describes a method for characterizing the members of acombinatorial library, which method utilizes affinity selection incombination with mass spectrometry. Specifically, the members of thelibrary are brought into contact with a domain of interest to allow forbinding, i.e., the formation of a complex. After binding, the complex isseparated from the unbound members of the library, typically by washingthe unbound members from the column containing the complexes. Thecomplexes are then treated to elute the bound library components and theeluted components are analyzed by mass spectrometry. The elution methodsdescribed include the use of displacers, chaotrope agents, pH elution,salt gradients, temperature gradients, organic solvents, selectivedenaturants and detergents. Using such methods, the weakly bound membersof the library are purportedly eluted first and analyzed by massspectrometry, followed by the elution of the more strongly boundmembers.

There are several disadvantages associated with the “capture andrelease” methods for screening compound libraries that have beenpreviously reported. First, the procedure used to “release” the boundligands from the ligand-receptor complexes may alter the binding profilefor the various bound ligands, resulting in a false indication ofbinding strength. For example, using a pH gradient to release the boundmembers of the library may change the electronic character of thebinding site on the receptor causing ligands which are strongly boundunder physiological conditions to be prematurely released. Thus, thecharacterization of binding strength for various ligands based on theirrelative time of release may be misleading if the release conditions aredifferent from the binding conditions. Accordingly, these methods maynot accurately identify the most active members of a compound library.Additionally, certain conditions used for compound release, such as pHgradients, may irreversibly denature the receptor thus preventing itssubsequent use for screening compound libraries.

Additionally, when “capture and release” methods are employed, eachbound ligand is typically released over a relatively short period oftime resulting, for example, in an elution peak or “spike” for eachligand. Accordingly, the effluent produced using such methods istypically monitored continually, for example, by mass spectrometry sothat any particular elution peak is not missed. Thus, the number ofanalyses that can be conducted using any particular mass spectrometer islimited. Accordingly, it would be desirable to develop methods andapparatus for screening compound libraries that do not rely upon“capture and release” methodologies.

SUMMARY OF THE INVENTION

This invention is directed to apparatus for screening compoundlibraries. The compound libraries may be generated or obtained by anymeans including, by way of example, combinatorial chemistry techniquesor from fermentation broths, plant extracts, cellular extracts and thelike. The apparatus of this invention employ frontal chromatography (FC)in combination with mass spectrometry (MS) to screen the library ofcompounds to identify and rank those members of the library that bind toa target receptor.

In frontal chromatography, a target receptor is typically immobilized ona suitable solid support material and packed in a column. A mixturecontaining putative ligands is then continuously infused through thecolumn. Ligands having an affinity for the target receptor bind to thecolumn, but eventually the capacity of the column for each ligand isexceeded and the ligands elute or “break through” at their infusionconcentration. Once a ligand begins eluting from the column, it iscontinually present in the effluent. Compounds having little or noaffinity for the target receptor break through earlier in the effluentcompared to ligands having a higher affinity for the receptor.

In the present invention, mass spectrometry (MS) is employed tocontinuously or intermittently monitor the FC effluent. Using MS, theidentity and break through time for each ligand on the column can bedetermined. Thus, FC-MS allows the relative affinity of each member ofthe library for the target receptor to be determined relative to othermembers of the library under ligand-receptor binding conditions. Usingthe present apparatus, an accurate ranking of the relative affinity ofeach member of the compound library for the target receptor can beascertained.

Accordingly, in one of its apparatus aspects, the present invention isdirected to an apparatus for screening a compound library to determinethe relative or absolute affinity of a plurality of putative ligands toa target receptor or a plurality of target receptors, which apparatuscomprises:

(a) a column comprising a target receptor or a plurality of targetreceptors, each target receptor optionally attached to a solid phasesupport, and having a inflow end and an outflow end, wherein said columnis capable of having a compound library comprising a plurality ofputative ligands applied thereto under frontal chromatography conditionsto produce an effluent from the outflow end of the column;

(b) a first reservoir connected to the inflow end of said column forapplying the compound library to the column;

(c) a mass spectrometer connected to the outflow end of said column forcontinuously or intermittently analyzing the effluent from the column.

In a preferred embodiment, the above described apparatus furthercomprises:

(d) a second reservoir connected to the inflow end of the column forapplying either (i) a mixture comprising the compound library, at leastone void marker compound and an indicator compound or a plurality ofindicators compounds, (ii) at least one void marker compound and anindicator compound or a plurality of indicator compounds, or (iii) abuffer solution to the column.

In another preferred embodiment, the above described apparatus furthercomprises:

(e) a third reservoir connected to the outflow end of the column forsupplying a supplemental diluent to the effluent before analysis by themass spectrometer.

Preferably, the column employed in this invention will have an internaldiameter (i.d.) ranging from about 10 μm to about 4.6 mm. Morepreferably, the internal diameter of the column will be in the range offrom about 100 μm to about 250 μm.

Preferably, the column will range in length from about 1 cm to about 30cm, more preferably from about 2 cm to about 20 cm.

Preferably, each target receptor is independently selected from thegroup consisting of proteins, including recombinant proteins,glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes,lectins, selecting, cell-adhesion molecules, toxins, bacterial pili,transport proteins, receptors involved in signal transduction orhormone-binding, hormones, antibodies, major histocompatabilitycomplexes, immunoglobulin superfamilies, cadherins, DNA or DNAfragments, RNA and RNA fragments, whole cells, cell fragments, tissues,bacteria, fungi, viruses, parasites, preons, and synthetic analogs orderivatives thereof.

Additionally, each target receptor is preferably bound to a solid phasesupport. More preferably, each target receptor is covalently bound tothe solid phase support or bound via biotin-avidin orbiotin-streptavidin binding.

Preferably, the solid phase support used in this invention is selectedfrom the group consisting of polymeric (resin) beads, polymeric gels,glass beads, silica chips, silica capillaries, agarose, diatomaceousearths and pulp.

The column employed in this invention preferably contains from about 1fmol to about 10 nmol of target receptor active sites; preferably, fromabout 1 pmol to about 10 nmol of target receptor active sites.

Preferably, the mass spectrometer employed in this invention is anelectrospray mass spectrometer.

Additionally, since FC-MS does not require constant effluent monitoring,a plurality of FC-MS analyses can be conducted simultaneously using asingle mass spectrometer to intermittently monitor each column.Furthermore, under FC conditions, since ligands are always present inthe effluent once they break through the column, the intermittentmonitoring of each column does not necessarily require monitoring theactual break through time for each ligand. Therefore, a plurality ofFC-MS analyses can be conducted simultaneously using a single massspectrometer to intermittently monitor each column.

Accordingly, in another of its apparatus aspects, this inventionprovides an apparatus for screening a plurality of compound libraries todetermine the relative or absolute affinity of a plurality of putativeligands in each library to a target receptor or a plurality of targetreceptors, which apparatus comprises:

(a) a plurality of columns each column comprising a target receptor or aplurality of target receptors, each target receptor optionally attachedto a solid phase support, and each column having a inflow end and anoutflow end, wherein each of said columns is capable of independentlyhaving a compound library comprising a plurality of putative ligandsapplied thereto under frontal chromatography conditions to produce aneffluent from the outflow end of the column;

(b) a plurality of first reservoirs each connected to the inflow end ofone of the columns for applying a compound library to the columns;

(c) a mass spectrometer connected to the outflow end of each of saidcolumns for intermittently analyzing the effluent from each of thecolumn.

In a preferred embodiment, the above described apparatus furthercomprises:

(d) a plurality of second reservoirs each connected to the inflow end ofone of the columns for applying either (i) a mixture comprising thecompound library, at least one void marker compound and an indicatorcompound or a plurality of indicator compounds, (ii) at least one voidmarker compound and an indicator compound or a plurality of indicatorcompounds, or (iii) a buffer solution to the column.

In another preferred embodiment, the above described apparatus furthercomprises:

(e) a third reservoir connected to the outflow end of each of thecolumns for supplying a supplemental diluent to the effluent from eachcolumn before analysis by the mass spectrometer.

Preferably, the above described apparatus comprises from 2 to about 100columns, more preferably from 3 to about 50 columns; and still morepreferably from 5 to about 10 columns.

Preferably, each column is intermittently monitored for a period ofabout 0.5 seconds to about 10 seconds, preferably for about 1 second toabout 5 seconds, before switching to the next column.

The apparatus of this invention can also be employed to screen a targetreceptor or a plurality of target receptors for affinity to animmobilized ligand or plurality of ligands. This embodiment isparticularly useful for target validation studies on ligands havingbiological effects. Accordingly, in another of its apparatus aspects,this invention provides an apparatus for screening a target receptor ora plurality of target receptors to determine the relative affinity ofthe receptor or receptors to an immobilized ligand or ligands relativeto an indicator compound or a plurality of indicator compounds, whichapparatus comprises:

(a) a column comprising a ligand or a plurality of ligands wherein eachligand is bound to a solid phase support, said column having a inflowend and an outflow end and further wherein said column is capable ofhaving a target receptor or a plurality of target receptors appliedthereto under frontal chromatography conditions to produce an effluentfrom the outflow end of the column;

(b) a first reservoir connected to the inflow end of said column forapplying the target receptor or receptors to the column;

(c) a second reservoir connected to the inflow end of the column forapplying either (i) a mixture comprising the target receptor orreceptors, at least one void marker compound and an indicator compoundor a plurality of indicators compounds, (ii) at least one void markercompound and an indicator compound or a plurality of indicatorcompounds, or (iii) a buffer solution to the column.

(d) a mass spectrometer connected to the outflow end of said column forcontinuously or intermittently analyzing the effluent from the column.

In a preferred embodiment, the above apparatus further comprises:

(e) a third reservoir connected to the outflow end of the column forsupplying a supplemental diluent to the effluent before analysis by themass spectrometer.

In a preferred embodiment, each ligand employed in the above apparatusis selected from the group consisting of carbohydrates, monosaccharides,oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides,polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,polynucleotides, lipids, retinoids, steroids, glycopeptides,glycoproteins, glycolipids, proteoglycans, and synthetic analogs orderivatives thereof.

In another preferred embodiment, each ligand is selected from the groupconsisting of synthetic small molecule organic compounds.

A plurality of such FC-MS analyses can also be conducted simultaneouslyusing a single mass spectrometer to intermittently monitor each column.Accordingly, in yet another of its apparatus aspects, the presentinvention provides an apparatus for screening a plurality of targetreceptors to determine the relative affinity of the receptors to animmobilized ligand or ligands relative to an indicator compound or aplurality of indicator compounds, which apparatus comprises:

(a) a plurality of columns each column comprising a ligand or aplurality of ligands wherein each ligand is bound to a solid phasesupport, and each column having a inflow end and an outflow end, whereineach of said columns is capable of independently having a targetreceptor or a plurality of target receptors applied thereto underfrontal chromatography conditions to produce an effluent from theoutflow end of the column;

(b) a plurality of first reservoirs each connected to the inflow end ofone of the columns for applying a target receptor or a plurality oftarget receptors to the columns;

(c) a plurality of second reservoirs each connected to the inflow end ofone of the columns for applying either (i) a mixture comprising thetarget receptor or plurality of target receptors, at least one voidmarker compound and an indicator compound or a plurality of indicatorcompounds, (ii) at least one void marker compound and an indicatorcompound or a plurality of indicator compounds, or (iii) a buffersolution to the column.

(d) a mass spectrometer connected to the outflow end of each of saidcolumns for intermittently analyzing the effluent from each of thecolumn.

In a preferred embodiment, the above apparatus further comprises:

(e) a third reservoir connected to the outflow end of each of thecolumns for supplying a supplemental diluent to the effluent from eachcolumn before analysis by the mass spectrometer.

Preferably, the above described apparatus comprises from 2 to about 100columns, more preferably from 3 to about 50 columns; and still morepreferably from 5 to about 10 columns.

Preferably, each column is intermittently monitored for a period ofabout 0.5 seconds to about 10 seconds, preferably for about 1 second toabout 5 seconds, before switching to the next column.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative apparatus for screening compoundlibraries using frontal chromatography in combination with a massspectrometer.

FIG. 2 illustrates a representative apparatus for screening compoundlibraries using a plurality of frontal chromatography columns incombination with a mass spectrometer.

FIG. 3 illustrates another representative apparatus for screeningcompound libraries using a plurality of frontal chromatography columnsin combination with a mass spectrometer.

FIG. 4 illustrates a representative apparatus for sequentially screeningcompound libraries with an indicator compound using a plurality offrontal chromatography columns in combination with a mass spectrometer.

FIG. 5A shows a total ion chromatogram (TIC) from a FC-MS run using sixrepresentative oligosaccharides having varying affinity for acarbohydrate-binding antibody that recognizes the3,6-dideoxy-D-galactose (abequose) epitope in Salmonella paratyphi BO-antigens.

FIG. 5B shows selected ion chromatograms for the six oligosaccharidesreconstructed from the TIC shown in FIG. 5A.

FIGS. 5C, 5D and 5E show mass spectra generated from time-slices of theTIC shown in FIG. 5A.

FIG. 6 shows a plot of ([A]₀(V-V₀))⁻¹ versus [A]₀ ⁻¹ forαGal(1→2)[αAbe(1→3)]αMan-OCH₃.

FIG. 7A shows a selected ion chromatogram from a FC-MS run using anindicator compound in the absence of a compound library.

FIG. 7B shows a selected ion chromatogram from a FC-MS run using anindicator compound in the presence of a compound library.

FIG. 8 shows a selected ion chromatogram from a FC-MS run using fourrepresentative oligosaccharides having varying affinity for choleratoxin B subunit.

FIG. 9 shows a selected ion chromatogram from a FC-MS run using asynthetically prepared GM₁ analog.

FIG. 10 illustrates the “roll-up” effect in a selected ion chromatogramfrom a FC-MS run using an indicator compound in the presence of acompound library.

FIG. 11 is a graph of the reduction of column activity as a function oftime for two different compound libraries, the first library containingmany weak binders and the second library containing strong binders.

FIG. 12A shows schematically the synthesis of a compound librarycontaining 100 tripeptides. FIG. 12B shows an electrospray mass spectrumof the compound library.

FIG. 13 shows a chromatogram of three infusion/wash cycles of a compoundlibrary containing 100 tripeptides, an indicator compound (dashed line)and a void marker compound (solid line).

FIG. 14A illustrates the V-V₀ value for an indicator compound (dashedline) relative to a void marker compound (solid line) immediately beforeequilibration of a column with a compound library containing 100tripeptides. FIG. 14B illustrates the V-V₀ value for the indicatorcompound immediately after equilibration of the column with the compoundlibrary.

FIG. 15A shows a total ion chromatogram for a compound librarycontaining 100 tripeptides. FIG. 15B shows a selected ion chromatogramfor m/z 419.2.

FIG. 16A shows a selected ion chromatogram for fPR. FIG. 16B shows aselected ion chromatogram of fPR and fPR-chloromethyl ketone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides apparatus for screening compoundlibraries using frontal chromatography in combination with massspectrometry. When describing the apparatus of this invention, thefollowing terms have the following meanings, unless otherwise indicated.All terms not defined herein have their conventional art-recognizedmeaning.

Definitions

The term “buffer” refers to a solution that stabilizes the bindingactivity of the target receptor. Typical buffers include, by way ofillustration, pH buffers and buffers containing specific molecules,either organic or inorganic, required to stabilize the binding activityof a specific target receptor.

The term “break through time” refers to the period of time betweenelution of the void volume and the front corresponding to the elution ofa particular compound during frontal chromatography. The term “breakthrough curve” refers to the signal intensity as a function of timeresulting from the infusion of compound(s) through a column underfrontal chromatography conditions. Typically, the break through curve iscomprised of a front (fast-rising section) and a plateau (horizontalsection).

The term “compound library” refers to a mixture or collection of one ormore putative ligands generated or obtained in any manner. Preferably,the library contains more than one putative ligand or member.

The term “electrospray” refers to the generation of gas-phase ions froma flowing solution. Electrospray is typically performed at atmosphericpressure in an electric field with or without assisted nebulization andsolvent evaporation.

The term “effluent” refers to the solvent or solution emerging orexiting from the frontal chromatography column.

The term “frontal chromatography conditions” refers to chromatographyconditions in which a solution of compounds, such as putative ligandsand/or indicator compounds, is applied or infused through a column togenerate a break through curve. Typically, under frontal chromatographyconditions, putative ligands are infused continuously at a constantconcentration through a column containing a target receptor such thatthe target receptor is continuously contacted with the putative ligandsduring the chromatography.

The term “indicator compound” or “indicator” refers to a compound havinga known affinity or specificity for the target receptor and a measurablebreak through time under frontal chromatography conditions. Whenscreening target receptors for affinity to a particular ligand(s), theindicator may also be a compound, such as a protein, or other biologicalentity, such as a cell or cell fragment, having a known affinity orspecificity for the ligand(s). The break through time for the indicatoris typically referenced to the void volume of the system.

The term “ligand” refers to a molecule or group of molecules that bindto one or more specific sites of a receptor. Representative ligandsinclude, by way of illustration, carbohydrates, monosaccharides,oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides,polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,polynucleotides, including DNA and DNA fragments, RNA and RNA fragmentsand the like, lipids, retinoids, steroids, glycopeptides, glycoproteins,glycolipids, proteoglycans and the like, and synthetic analogues orderivatives thereof, including peptidomimetics, natural products ornaturally-occurring small molecule organic compounds (i.e., compoundsproduced by and/or isolated from natural sources, such as soil, water,cells, plants, fungi, animals and the like), synthetic small moleculeorganic compounds, inorganic ions, organometallic compounds and thelike, and mixtures thereof. The term “putative ligand” refers to aligand whose affinity or specificity for a target receptor, if any, hasnot been determined.

The term “microcolumn” refers to a column having an internal diameterless than or equal to about 1 mm.

The term “natural products” refers to compounds isolated from naturalsources, such as cells, plants, fungi, animals and the like. The term“naturally-occurring small molecule organic compounds” refers to naturalproducts that are organic compounds generally having a molecular weightless than about 1000, preferably less than about 500.

The term “selected ion chromatogram” refers to a plot of ion abundancevs. time constructed from the intensity of a single ion. A selected ionchromatogram can be prepared from a scan or selected ion monitoringmode.

The term “selected ion monitoring” refers to the detection of a fewpre-selected ions using a mass spectrometer (e.g. quadrupoles).

The term “signal intensity” refers to the measured response of aninstrument to a stimulus, for example, the current output of a highenergy dynode detector resulting from the impact of an ion.

The term “solid support” or “solid phase support” refers to an inertmaterial or molecule to which a target receptor may be bound or coupled,either directly or through a linking arm.

The term “synthetic small molecule organic compounds” refers to organiccompounds generally having a molecular weight less than about 1000,preferably less than about 500, which are prepared by synthetic organictechniques, such as by combinatorial chemistry techniques.

The term “supplemental diluent” or “make-up flow” refers to a solutionor solvent which is combined with the effluent from a column before theeffluent passes through the mass analyzer of a mass spectrometer.

The term “target receptor” or “receptor” refers to a molecule or a groupof molecules capable of binding a ligand at a specific site.Representative examples of target receptors include, by way of example,proteins, including recombinant proteins, glycoproteins,glycosaminoglycans, proteoglycans, integrins, enzymes, lectins,selectins, cell-adhesion molecules, toxins, bacterial pili, transportproteins, receptors involved in signal transduction or hormone-binding,hormones, antibodies, major histocompatability complexes, immunoglobulinsuperfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments,whole cells, cell fragments, tissues, bacteria, fungi, viruses,parasites, preons, and synthetic analogs or derivatives thereof.

The term “target receptor active site” refers to the binding site ofinterest on a particular target receptor.

The term “total ion chromatogram” refers to a plot of ion abundance vs.time constructed from a summation of all ion intensities in a scan. In atotal ion chromatogram, the number of scans are linearly related totime.

The term “void marker compound” or “void marker” refers to a compoundthat elutes from column at the void volume. The void marker compound isused to identify the void volume of a column used under frontalchromatography conditions.

The term “void volume” or “V₀” refers to the volume of solution whichpasses through a frontal chromatography column from the point ofinfusion to the point of detection of a compound, i.e. a putativeligand, in the absence (or suppression) of a binding event. Since theflow rate is typically constant, void volume is generally specified interms of a retention time for the compound. Putative ligands having noaffinity for the target receptor typically elute from column at the voidvolume.

The compound libraries employed in this invention may be prepared orobtained by any means including, but not limited to, combinatorialchemistry techniques, fermentation methods, plant and cellularextraction procedures and the like. Methods for making combinatoriallibraries are well-known in the art. See, for example, E. R. Felder,Chimia 1994, 48, 512-541; Gallop et al., J. Med. Chem. 1994, 37,1233-1251; R. A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten etal., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84;Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectivesin Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992,89, 5381-5383; Gordon et al., J. Med. Chem. 1994, 37, 1385-1401; Lebl etal., Biopolymers 1995, 37 177-198; and references cited therein. Each ofthese references is incorporated herein by reference in its entirety.

Any type of molecule that is capable of binding to a target receptor maybe present in the compound library. For example, compound librariesscreened using this invention may contain naturally-occurring molecules,such as carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, peptides, oligopeptides, polypeptides,proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides,including DNA and DNA fragments, RNA and RNA fragments and the like,lipids, retinoids, steroids, glycopeptides, glycoproteins, glycolipids,proteoglycans and the like; or analogs or derivatives ofnaturally-occurring molecules, such peptidomimetics and the like; andnon-naturally occurring molecules, such as “small molecule” organiccompounds generated, for example, using combinatorial chemistrytechniques; organometallic compounds, inorganic ions, and mixturesthereof. The term “small molecule organic compound” refers to organiccompounds generally having a molecular weight less than about 1000,preferably less than about 500.

A particular advantage of FC-MS is that compound libraries containingisomers may be screened to determine, for example, if only one isomer(e.g. an enantiomer or diastereomer) is binding to the target receptor,or if the isomers have different affinities for the target receptor. Inthis regard, if the isomers have different affinities for the targetreceptor, a different break through time will be observed for eachisomer.

The compound libraries employed in this invention will typically containa plurality of members or putative ligands. When a indicator compound isemployed, the compound library will preferably contain less than about50,000 members, more preferably, the compound library will contain lessthan about 10,000 members. When an indicator compound is not employed,the compound library will preferably contain less than about 10,000members; more preferably, from 1 to about 1,000 members; and still morepreferably, from about 5 to about 100 members.

The present apparatus is useful for analyzing the affinity of members ofa compound library for any target receptor or domain which binds orcomplexes with a ligand. For example, the target receptor may beselected from, but is not limited to, proteins, including recombinantproteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins,enzymes, lectins, selectins, cell-adhesion molecules, toxins, bacterialpili, transport proteins, receptors involved in signal transduction orhormone-binding, hormones, antibodies, major histocompatabilitycomplexes, immunoglobulin superfamilies, cadherins, DNA or DNAfragments, RNA and RNA fragments, whole cells, cell fragments, tissues,bacteria, fungi, viruses, parasites, preons, and synthetic analogs orderivatives of any of the above. If desired, more than one targetreceptor may be employed when screening a compound library using themethods of this invention.

When employing the apparatus of this invention, the target receptor (ora ligand) is optionally bound or coupled to a solid support. Preferably,the target receptor is covalently bound or coupled to the solid support.However, in some cases, such as when whole cells or organisms areemployed as the target receptor, the cells or organisms may be containedwithin the column by using, for example, a porous frit or membrane atthe outflow end of the column. Supports for receptors are well-known inthe art and many are commercially available. Any such conventionalsupport may be used in this invention. Representative supports include,by way of illustration, polymeric (resin) beads, polymeric gels, glassbeads, silica chips and capillaries, agarose, diatomaceous earths, pulp,and the like. When silica capillaries are used as the solid support, thetarget receptor is bound directly to the walls of the column. Preferredsolid supports for use in this invention are those having minimalnon-specific binding properties. A preferred solid support isderivatized porous polystyrene-divinylbenzene polymer beads, such asPOROS beads (available from Perseptive Biosystems, Framingham, Mass.). Aparticularly preferred solid support is silica particles, such ascontrolled pore glass (available from CPG Inc., Lincoln Park, N.J.).

The target receptor (or ligand) can be bound or coupled to the supportusing any art-recognized procedure. For example, the target receptor canbe bound using direct immobilization techniques (i.e., covalent bindingvia a sulfihydryl, amino or carboxyl group and the like), covalentbinding through a linking or spacer arm, biotin-avidin binding,biotin-streptavidin binding, antibody binding such as antibody-protein Abinding, GST-glutathione binding, ion exchange absorption, hydrophobicinteraction, expression of the target receptor as a recombinant proteinfused to maltose binding protein, fusion of the target receptor with apeptide which binds selectively to an affinity column, and the like.Such methods are well-known in the art and kits for practicing many ofthese methods are commercially available. See, for example, Stammers etal., FEBS Lett. 1991, 283, 298-302; Herman et al., Anal. Biochemistry1986, 156, 48; Smith et al., FEBS Lett. 1987, 215, 305; Kilmartin etal., J. Cell. Biol. 1982, 93, 576-582; Skinner et al., J. Biol. Chem.1991, 266, 14163-14166; Hopp et al., Bio/Technology 1988, 6, 1204-1210;H. M. Sassenfeld, TIBTECH 1990, 8, 88-93; Hanke et al., J. GeneralVirology 1992, 73, 654-660; Ellison et al., J. Biol. Chem. 1991, 267,21150-21157; U. K. Pati, Gene 1992, 114, 285-288; Wadzinski et al., J.Biol Chem. 1992, 267, 16883-16888; Field et al., Mol. Cell. Biol. 1988,8, 2159-2165; Gerard et al., Biochemistry 1990, 29, 9274-9281;Ausselbergs et al., Fibrinolysis 1993, 7, 1-13; Hopp et al.,Biotechnology 1988, 6, 1205-1210; Blanar et al., Science 1992, 256,1014-1018; Lin et al., J. Org. Chem. 1991, 56, 6850-6856; Zastrow etal., J. Biol. Chem. 1992, 267, 3530-3538; Lim et al., J. InfectiousDisease 1990, 162, 1263-1269; Goldstein et al., Virology 1992, 190,889-893; and the articles in IBI FLAG Epitope Vol. 1: No. 1, September1992; and references cited therein. Each of these references isincorporated herein by reference in its entirety.

In a preferred embodiment of this invention, the target receptor isbound or coupled to the solid support using biotin-avidin,biotin-streptavidin or a related-type binding. In this procedure, thetarget receptor is typically biotinylated with a biotin reagentcontaining a spacer arm. The biotinylated target receptor is thencontacted with an avidin-containing solid support. The resultingbiotin-avidin complex binds the target receptor to the solid support.

Procedures for biotinylating biomolecules are well-known in the art andvarious biotin reagents are commercially available. See, for example, E.A. Bayer et al., Meth. Enzymol. 1990, 184, 51; U. Bickel et al.,Bioconj. Chem. 1995, 6, 211; H. Hagiwara et al., J. Chromatog. 1992,597, 331; “Avidin-Biotin Chemistry Handbook” (available from Pierce,Rockford, Ill., Catalog Item No. 15055) and references cited therein. Apreferred biotin reagent is NHS-LC-biotin (available from Pierce). Theextent of biotin incorporation using such reagents can be monitored by,for example, matrix-assisted laser desorption/ionization as described inD. C. Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387, or by otherart-recognized methods as described in the “Avidin-Biotin ChemistryHandbook” (Pierce). Preferably, an average of about 1 to about 50biotins are incorporated per target receptor, more preferably about 1 toabout 10 biotins per target receptor.

The biotinylated target receptor is typically coupled with an avidin- orstreptavidin-containing solid support or related material. Such supportsare commercially available or can be prepared by art-recognizedprocedures. Preferred avidin-containing supports includeUltralink-immobilized avidin (available from Pierce) and POROS 20immobilized streptavidin (available from Perseptive Biosystems). Thebiotinylated target receptor is typically coupled with theavidin-containing support by contacting the receptor with the support ina suitable buffer, such as phosphate buffered saline (pH 7), for about0.5 to 4 hours at a temperature ranging from about 4° C. to about 37° C.Preferably, after coupling the biotinylated target receptor to theavidin-containing support, any remaining avidin binding sites on thesupport are blocked by contacting the solid support with an excess offree biotin.

The target receptor may be bound or coupled to the solid support eitherprior to or after introducing the solid support material into a column.For example, the biotinylated target receptor may be contacted orincubated with the avidin- or streptavidin-containing solid support andthe resulting solid support containing the target receptor subsequentlyintroduced into a column. Alternatively, the avidin- orstreptavidin-containing solid support can be first introduced into thecolumn and the biotinylated target receptor then cycled through thecolumn to form the solid support containing the target receptor in thecolumn. Either of these methods may also be used with any of the otherpreviously mentioned procedures for coupling the target receptor to thesolid support.

When more than one target receptor is employed, each target receptor canbe bound to the same solid support using the procedures describedherein. Alternatively, each target receptor can be bound to a separatesolid support and the solid support materials containing the targetreceptors subsequently homogenized and packed into the column.

The solid support material may be introduced into the column using anyconventional procedure. Typically, the solid support is slurried in asuitable diluent and the resulting slurry is pressure packed or pumpedinto the column. Suitable diluents include, by way of example, bufferssuch as phosphate buffered saline (PBS) solutions, preferably containinga preservative such as sodium azide, and the like.

Generally, the activity of the target receptor will determine the sizeof the column employed in this invention, i.e., a smaller column volumemay be employed when the target receptor has more activity per unitcolumn volume. Typically, the column employed in this invention willhave an internal diameter (i.d.) ranging from about 10 μm to about 4.6mm. Preferably, the internal diameter of the column will be in the rangeof from about 100 μm to about 250 μm. The column will typically range inlength from about 1 cm to about 30 cm, preferably from about 2 cm toabout 20 cm. Preferably, the column will have from about 1 fmol to about10 nmol of target receptor active sites per column; more preferably,from about 0.1 pmol to about 10 nmol of target receptor active sites percolumn; still more preferably, from about 0.1 pmol to about 100 pmol oftarget receptor active sites per column.

If an indicator compound is employed, the length of the column and itsi.d. will also depend upon the K_(d) of the indicator compound (i.e., asmaller column may be used when the indicator has a higher affinity forthe target receptor). Preferably, when an indicator is employed, thecolumn length and i.d. are selected so that the indicator compoundelutes a measurable quantity after the void volume.

The body of the column employed in this invention may be comprised ofany conventional column body material including, by way of illustration,poly(ether ether ketone) (PEEK), fused silica, silicon microchips,stainless steel, nylon, polyethylene, polytetrafluoroethylene (Teflon)and the like. Preferably, the column body is comprised of poly(etherether ketone).

Alternatively, the column may be open-faced, such as in thin-layerchromatography (TLC) plate configuration or a gel plate. In thisembodiment, the effluent can be analyzed using the electrospraytechniques described herein. Alternatively, the plate or plate-likeconfiguration can be subjected to matrix-assisted laserdesorption/ionization (MALDI) analysis at any stage of thechromatography to provide a molecular weight analysis for a plurality ofpositions on the plate.

After the solid support containing the target receptor is introduced orformed in the column, the column is typically flushed with a suitablediluent to remove any unbound target receptor or impurities. Suitablediluents for flushing the column include, for example, phosphatebuffered saline, TRIS buffers and the like. If desired, a detergent mayalso be included in the buffer to facilitate removal of unbound targetreceptor or impurities.

After the column is flushed, the column is typically equilibrated with abuffer suitable for frontal chromatography and compatible with massspectrometry. Volatile buffers are generally preferred for use with massspectrometry. For frontal chromatography, a buffer is typically selectedto promote receptor-ligand interaction. Suitable buffers for use inFC-MS include, by way of example, ammonium acetate, ammonium formate andthe like.

Procedures for conducting frontal chromatography are well-known in theart. See, for example, K.-I. Kasai et al., Journal of Chromatography1986, 376, 33-47; D. S. Hage et al., Journal of Chromatography B, 1997,669, 449-525 and references cited therein. The disclosures of thesereferences are incorporated herein by reference in their entirety.Typically, the compound library is continuously applied or infused intothe column containing the target receptor. Under these conditions, thetarget receptor is continuously contacted or challenged with each of themembers of the compound library. The column is driven to dynamicequilibrium by continuously applying the compound library. The column isdriven to dynamic equilibrium by continuously applying the compoundlibrary to the column. Library members having different bindingconstants to the target receptor display different break through timesor hold-up volumes on the column, i.e. those members having a higheraffinity for the target ligand have a longer break through time on thecolumn or a larger hold-up volume until they begin to elute from orbreak-through the column at their initial infusion concentration. Unlikezonal chromatographic methods, no physical separation of the librarymembers is achieved using frontal chromatography. Suitable methods forconducting FC-MS are described in U.S. patent application Ser. No.09/390,694, filed Dec. 28, 1998, U.S. Pat. application Ser. No.09/276,444, filed Mar. 25, 1999 and its equivalent, Patent CooperationTreaty (“PCT”) application No. PCT/CA99/00266 filed Mar. 26, 1999, thedisclosures of which are incorporated herein by reference in theirentirety.

During the frontal chromatography, the column is typically at atemperature in range from about 0° C. to about 90° C.; preferably fromabout 4° C. to about 60° C.; more preferably from about 20° C. to about40° C.

When a ligand has a very slow on-rate, it may be desirable to conductthe column equilibrium over an extended period of time. The column canbe equilibrated by infusing the compound library through the column fora period sufficient to allow the column to reach equilibrium. Forexample, this can be achieved by increasing the equilibration time,i.e., to about 0.25 to 24 hours; or by reducing the flow rate to about1% to 10% of the usual flow rate. Alternatively, a sequence of stop-flowcycles may also be conducted.

In the apparatus of this invention, a mass spectrometer is coupled tothe column to analyze the effluent. Mass spectrometry is particularlyuseful in the present invention since it allows for both detection andidentification of the library members present in the effluent. In thisregard, mass spectrometry allows the eluting members of the library tobe identified based on their mass/charge ratio.

Prior to analyzing the effluent from the column by mass spectrometry,the effluent is optionally diluted with a supplemental diluent or“make-up flow” and the combined flow is directed into, for example, theelectrospray mass spectrometer. Typically, the supplemental diluentcomprises a major amount of an organic solvent and a minor amount of anaqueous buffer. The organic solvent is selected so as to promote astable and efficient electrospray. Representative organic solventssuitable for use in the supplemental diluent include, by way of example,acetonitrile, methanol, isopropanol and the like. A preferred organicsolvent is acetonitrile. Typically, the amount of supplemental diluentemployed is adjusted so that the combined flow rate of the effluent andthe supplemental diluent is less than about 100 μL/min. Preferably, thecombined flow rate entering the mass spectrometer ranges from about 100nL/min to about 20 μL/min.

Methods for analyzing effluents using mass spectrometry are well-knownin the art. Any type of mass spectrometry which is capable of directlyor indirectly analyzing the components present in a solution may beemployed in this invention including, for example, electrospray massspectrometry (ES-MS), atmospheric pressure chemical ionization (APCI),membrane introduction mass spectrometry (MIMS), continuous flow fastatom bombardment (cf-FAB), thermospray techniques, particle beam, movingbelt interfaces and the like. Electrospray mass spectrometry isparticularly preferred. Apparatus and techniques for conductingelectrospray mass spectrometric analysis are described, for example, inS. J. Gaskell, “Electrospray: Principles and Practice” J. Mass.Spectrom. 1997, 32, 677-688 and reference cited therein. When theeffluent is collected and optionally pre-treated prior to mass spectralanalysis, any of the above described ionization methods may be used aswell as MALDI, fast atom bombardment (FAB), massive cluster impact,electron impact, chemical ionization, secondary ion mass spec and fielddesorption ionization techniques.

The mass spectrometer employed in the methods of this invention may beof any type (i.e., scanning or dynamic) including, by way ofillustration, quadrupole, time of flight, ion trap, FTICR and the like.Typically, the mass spectrometer parameters are set to provide thehighest sensitivity for the eluting compounds. Generally, when anelectrospray mass spectrometer is employed, such adjustments willinvolve optimization of, for example, nebulizer pressure, drying gasflow rate, ion transmission and electrospray needle position. Forexample, the nebulizer pressure will typically range from about 0 psi toabout 60 psi; and the drying gas flow rate will range from about 0 L/minto about 50 L/min. A total ion chromatogram is typically measured andmonitored in real-time. The size of the column, the concentration of thecompound library and the flow rate will generally determine therun-time. Typical run times range from about 1 min to about 60 min.

Upon completion of the frontal chromatography, the column is optionallyregenerated by washing with a large volume of the binding buffer, withor without a competitive ligand. In this regard, a particular advantageof the present method is that denaturing of the target receptor is notrequired at any point in the procedure. Accordingly, columns may bere-used many times generally with no observable loss of activity orleaching of the target receptor. Alternatively, since the methods ofthis invention employ very small amounts of target receptor, the columnmay be disposed of after a single use.

A representative apparatus for conducting the screening methods of thisinvention is illustrated in FIG. 1. As shown in FIG. 1, a firstreservoir 1, containing a buffer solution, and a second reservoir 2,containing a solution of a compound library in a buffer, are connectedvia tubing 3 to valve 4. In FIG. 1, reservoirs 1 and 2 are syringesalthough any similar reservoir may be employed. Valve 4 allows thesolutions from reservoirs 1 or 2 to be directed into a waste container 5or into the inflow end of column 6. Column 6 contains the targetreceptor bound to a solid phase support, the column wall or otherwiseretained within the column. The outflow end of column 6 is connected toa mixing tee 7, which is also connected to reservoir 8, containing asupplemental diluent, via tubing 9. The effluent from column 6 is mixedwith the supplemental diluent from reservoir 8 in mixing tee 7 and theoutflow is directed via tubing 10 to an electrospray mass spectrometer11. To control the flow from reservoirs 1, 2 and 8, pressure is appliedto plungers 12 via, for example, a pump. A simpler manifestation wouldinclude just the column 6 connected to the reservoir 2, with the outflowdirected via tubing 10 to an electrospray mass spectrometer.Alternatively, a configuration involving an HPLC pump and a valve withan oversized injection loop for the library solution could be used, suchan apparatus is described, for example, in E. Breklan et al., Biochem.1996, 35, 12141-12145.

In another of its embodiments, the apparatus of this invention can beused for screening a compound library to determine if any member of thelibrary has an affinity for a target receptor that interferes with thebinding of a pre-selected indicator compound or a mixture of indicatorcompounds. In this embodiment, the break through time of an indicatorcompound having a known affinity for the target receptor is determinedafter the column has been equilibrated with the compound library andcompared to the break through time for the indicator compound in theabsence of the compound library. If the indicator compound has a shorterbreak through time after equilibration with the compound library, thecompound library contains one or more ligands having an overall affinityfor the target ligand which is higher than the indicator compound. Sincean indicator compound can be selected having a relatively short breakthrough time on the column, a significant advantage of this embodimentis that compound libraries can be rapidly screened, e.g., in less than 5minutes, to identify those libraries having a pre-determined minimumlevel of affinity for the target receptor. When a library is identifiedas having the pre-determined minimum level of affinity for the targetreceptor, the library can be further analyzed using FC-MS to identifythe ligands binding to the target receptor.

One advantage of using an indicator compound is that the screening timefor each library is significantly reduced since only the indicatorcompound needs to be monitored relative to a void marker compound.Additionally, since the indicator compound binds to the target receptorat the active site of interest, a change in the break through time forthe indicator reflects an affective interaction of a member (or members)of the library with the target receptor. This interaction includes, byway of example, binding at the active site, and binding at a different ,non-overlapping site that affects the ability of the target receptor tobind the indicator compound. This method is particularly advantageous inthat nonspecific binding to the target receptor that does not alter theactive site will not cause a shift in the break through time for theindicator compound. Accordingly, non-specific binding of the library tothe target receptor does not provide false leads.

The indicator compound used in this embodiment of the invention istypically selected so as to have a relatively weak affinity for thetarget receptor. This permits the indicator compound to rapidly elute orbreak through the column, thus shortening the period of time necessaryto monitor the effluent. An indicator compound having a break throughtime on the column less than about 5 minutes in the absence of thecompound library is preferred. Alternatively, an indicator having astrong affinity for the target receptor may be used thereby allowingsmaller columns to be employed. When an indicator compound having astrong affinity is used, the compound library will typically be appliedto the column at a higher concentration. The break through time for theindicator compound on the column in the absence of the compound libraryis determined using the FC-MS procedures described herein. The affinityof the indicator compound for the target receptor can be determinedusing conventional techniques, such as microcalorimetry and the like; orby using the FC-MS methods of this invention. Preferably, the indicatorcompound will also have a unique mass in comparison to the members ofthe compound library so that the indicator compound can be unambiguouslyidentified by mass spectrometry. Generally, when using an indicatorcompound and a quadrupole mass spectrometer, only the m/z of theindicator compound and the compounds representing the void volume aremonitored to provide for a greater signal to noise ratio.

Representative examples of indicator compounds suitable for use withspecific target receptors include, by way of illustration,αAbe(1→3)αTal-OCH₃ (K_(d)=0.2 mM) for use with a monoclonal antibodythat recognizes the 3,6-dideoxy-D-galactose (abequose) epitope inSalmonella paratyphi B O-antigens; phytic acid (K_(d)=1 μM) for use withL-selectin, and the like. Additionally, more than one indicator compoundmay be employed. The indicator may also be coupled or conjugated toanother molecule containing an atom, isotope or molecular fragment whichfacilitates its detection. For example, the indicator compound can beconjugated to polyethylene glycols (PEGs) so that the mass spectra wouldcontain peaks differing by 44 units thereby facilitating detection ofthe of indicator compound.

The break through time for the indicator compound is typically measuredrelative to a void marker compound. The void marker compound is acompound which elutes from the column at the void volume. Preferably,the void marker compound is structurally similar to the indicatorcompound, but has no affinity for the target receptor of interest. Insome cases, putative ligands in the compound library which have noaffinity for the target receptor may serve as the void marker compounds.

When a functional target receptor, such as an enzyme, is employed inthis invention, the substrate for the functional target receptor may beused as the indicator. By doing so, the loss of function or inhibitionof the target receptor can be monitored in the presence of a compoundlibrary. In this embodiment, a first indicator compound is selectedwhich is a substrate for the functional target receptor, i.e. the firstindicator is a compound which is capable of being chemically modified bythe functional target receptor to produce a second indicator compound.Using the frontal chromatography procedures described herein, the firstindicator compound and the compound library to be analyzed are appliedto or infused into a column comprising the functional target receptor.The effluent from the column is then monitored for the presence and/orconcentration of the first and/or the second indicator compounds, i.e.,the substrate and/or the reaction product. An increase in the expectedconcentration of the first indicator compound or a decrease in theexpected concentration of the second indicator compound (as determinedby conducting the frontal chromatography of the indicator compounds inthe absence of the compound library) indicates that the functionaltarget receptor is being inhibited by one or more members of thecompound library. When a compound library is identified as having aninhibitor present in the library for the functional target receptor, thelibrary can be further analyzed using FC-MS to identify the ligandsbinding to the target receptor.

When using an indicator compound is employed, the break through time forthe indicator compound is first determined by applying the indicatorcompound and the void marker compound to the column containing thetarget receptor under frontal chromatography conditions. The column isthen typically equilibrated or partially equilibrated with the compoundlibrary to be screened. Generally, the compound library is applied orinfused into the column for a time sufficient to allow all of thelibrary members to break through the column. The effluent during thisperiod may be presented to the mass spectrometer for analysis or may becollected for recycling or disposal. Once the column has beenequilibrated or partially equilibrated with the compound library, amixture comprising the compound library, void marker compound and theindicator compound is applied to or infused into the column using thefrontal chromatography procedures described herein. Preferably, theindicator compound will be present in the mixture in a concentrationless than its K_(d) value. Typically, the indicator compound will bepresent in an amount ranging from about 1 nM to about 10 μM, morepreferably from about 10 nM to about 1 μM. The effluent from the columnis analyzed to determine the break through time for the indicatorcompound in the presence of the compound library and this time period iscompared to the pre-determined break through time for the indicatorcompound to ascertain whether the compound library has an affinity forthe target receptor.

Alternatively, the indicator compound and the void marker compound,without the compound library, can be applied or infused into the columnafter equilibration or partial equilibration of the column with thecompound library. This technique allows very strongly bound ligands orthose with slow off rates to be detected.

An indicator compound can also be useful in determining whether theoverall affinity of a compound library is due to the presence of aplurality of weak binders or to one or more strong binders. In thisembodiment, a column equilibration procedure is initiated by infusion ofthe compound library as described herein. The indicator compound, in thepresence of the library and the necessary void markers, is then passedthrough the column during the initial stages of the equilibrium process(typically at about 1 to about 5 minutes) and a first break through timefor the indicator compound is determined. The flow of compound librarywithout the indicator is then reestablished through the column. After aperiod of time, the indicator/library/void marker solution is againpassed through the column and a second break through time is determined.When the same indicator compound is used through this procedure, theintervening time period between each application of the indicatorcompound can be as short as the time necessary to wash off the indicatorcompound, and as long as the total equilibration time (e.g., 1 min to 60minutes, respectively). This cycle can be repeated any number of times.In this method, the discrimination between weak and strong bindersoccurs because a weak ligand will reach equilibrium on the column soonerthan a strong one. Monitoring of the column activity during theequilibration process is illustrated in FIG. 11 which is a graph of thereduction of column activity as a function of time for two differentlibraries: one containing many weak binders and one containing strongbinders. Even though the same overall reduction in column activity isachieved (as measured by the indicator compound), the rate of reductionis slower for the strong ligands compared to the weak ligands.

An alternative method for distinguishing between a plurality of weakbinders and one or more strong binders in a compound library isillustrated in FIG. 10. In this embodiment, an indicator compound(V_(n-1) in FIG. 10) is first selected having an affinity for the targetreceptor which is weaker than the putative ligands of interest (V_(n))but stronger than those not of interest (V_(1,max)). A mixturecomprising the compound library, the void marker compound and theindicator compound at a pre-determined initial concentration and signalintensity is then applied or infused into a column comprising the targetreceptor under frontal chromatography conditions to provide an effluent.In this embodiment, the column is not pre-equilibrated with the compoundlibrary. Preferably, the concentration of the indicator compound in themixture is greater than or equal to its dissociation constant for thetarget receptor. The effluent from the column is then analyzed by massspectrometry to determine a break through time for the indicatorcompound and its signal intensity. In the presence of a plurality ofweak binders (i.e., weaker than the indicator compound), the breakthrough time for the indicator will be less than its break through timein the absence of the compound library and the basic shape of the curvewill be unchanged. In the presence of one or more ligands having anaffinity for the target receptor greater than the indicator compound,the break through time will also be less than the break through time forthe indicator compound in the absence of the compound library, but theshape of the curve will also display a “roll up” effect as illustratedin FIG. 10. This “roll up” effect is due to the removal of boundindicator compound by the stronger ligand(s). Thus, for a short periodof time, the concentration of the indicator compound is higher than itsinfusion concentration until the stronger ligand(s) breaks through. Theamount of bound indicator compound removed is dependent upon the K_(d)value and the concentration of the stronger ligand(s) present in thelibrary. Weaker ligands do not exert this “roll up” effect because theypropagate through the column more quickly than the indicator compound.Detection or measurement of this “roll up” or bump in the break throughcurve (typically measured as a change in signal intensity) indicates thepresence of ligands in the compound library having an affinity for thetarget receptor greater than the indicator compound. The detection ormeasurement of a “roll up” effect may also be used when screening targetreceptors for affinity to an immobilized ligand(s).

In addition to detecting the indicator compound using mass spectrometry,other methods of detection may also be employed. Any detection methodthat can measure the indicator compound over the background signal ofthe library compounds can be used. For example, an indicator compoundcan be detected in the effluent from the column using, by way ofexample, fluorescence, infra-red absorption, UV-visible absorption,nuclear magnetic resonance (NMR), atomic spectroscopy (i.e., atomicadsorption spectroscopy (AAS), inductively coupled plasma-opticalemission spectroscopy (ICP-OES), etc.), flow cytometry, electrochemicaldetection and the like. Procedures and apparatus for detecting compoundsusing such methods are well-known in the art and any conventionalprocedures and apparatus may be used.

The apparatus of this invention allow a plurality of FC-MS analyses tobe conducted simultaneously using a single mass spectrometer tointermittently monitor each column. Unlike “capture and release” methodswhich typically provide an elution peak or “spike” for each ligand,FC-MS does not require constant effluent monitoring because once alibrary member breaks through the column, that member is continuouslypresent in the effluent and can be detected by the mass spectrometer.Therefore, a plurality of FC-MS analyses can be conducted simultaneouslyusing a single mass spectrometer to intermittently monitor each column.For example, using this invention, at least about 100 columns can beconducted simultaneously.

When employing multiple columns, each column is typically monitored fora brief period of time before switching to the next column. For example,with a quadrupole mass spectrometer, each column is typically monitoredsequentially for a period of about 0.5 seconds to about 10 seconds,preferably for about 1 second to about 5 seconds, before switching tothe next column. The effluent from each column is analyzed as describedherein using mass spectrometry. Generally, a single data file is used tocollect all of the data from the multiple column thereby generating acomposite total ion chromatogram. Subsequently, separate total ionchromatograms for each column are recreated by synchronizing columnswitching with mass spectrometry data acquisition.

In a preferred embodiment, each column will have an individualelectrospray needle for injection of the column's effluent into theelectrospray mass spectrometer. Any geometric arrangement of multipleelectrospray needles that allows for fast and repetitive sequences ofneedle advancement may be employed. A suitable apparatus for theinjection of multiple effluents into a electrospray mass spectrometer isdescribed in U.S. patent application Ser. No. 09/069,656, filed Apr. 29,1998, U.S. patent application Ser. No. 09/275,810, filed Mar. 25, 1999,and its equivalent, PCT application No. PCT/CA99/00264 filed Mar. 26,1999, the disclosures of which are incorporated herein in theirentirety. Alternatively, a linear moving row of electrospray needles(sprayers) and the like may be employed. See, for example, Q. Xue etal., Anal. Chem, 1997, 69, 426-430 and references cited therein, thedisclosed of which is incorporated herein by reference in its entirety.

A representative apparatus for screening compound libraries using aplurality of columns is illustrated in FIG. 2. As shown in FIG. 2, eachof a plurality of columns 13 is connected via tubing 14 and tee 15 to afirst reservoir 16, containing a solution of a compound library in abinding buffer, and a second reservoir 17, containing the bindingbuffer. In FIG. 2, reservoirs 16 and 17 are syringes although anysimilar reservoir may be employed. Each column 13 contains a targetreceptor bound to a solid phase support. The buffer solution inreservoir 17 is used to wash column 13 before or after introduction ofthe compound library. The outflow end of each column 13 is connected toa mixing tee 18, which is also connected to reservoir 19, containing asupplemental diluent, via tubing 20. The effluent from each column 13 ismixed with the supplemental diluent from reservoir 19 in mixing tees 18and the outflow is directed via tubing 20 and valves 21 into anelectrospray mass spectrometer 22, via an electronically-actuatedmulti-port selection valve 23, or into waste/recovery containers 24. Tocontrol the flow from reservoirs 16, 17 and 19, pressure is applied toplungers 25 via, for example, pumps.

Alternatively, in another embodiment illustrated in FIG. 3, the outflowfrom mixing tees 18 may be directed via tubing 20 into individualelectrospray needles 26 for mass spectrometer analysis.

When using an indicator compound, sequential runs of multiple columnsmay be advantageous since this allows the retention time for theindicator compound to be more accurately determined. Parallel infusionof the indicator through a plurality of columns is feasible provided anapparatus is used with a suitable high sampling rate (e.g. allowing fora minimum of five mass spectral measurements on the break through curveof the indicator for each of the columns. Such an apparatus is describedin U.S. patent application Ser. No. 09/069,656, filed Apr. 29, 1998,U.S. patent application Ser. No. 09/275,810, filed Mar. 25, 1999, andits equivalent, PCT application No. PCT/CA99/00264, filed Mar. 26, 1999.

A representative apparatus for sequentially screening compound librarieswith a indicator compound using a plurality of columns is illustrated inFIGS. 2, 3 and 4. The apparatus shown in FIGS. 2 and 3 are preferred andare employed as described herein for FC/MS. However, reservoir 16optionally contains a solution of the compound library plus theindicator compound and void marker compounds in a binding buffer, whilereservoir 17 optionally contains a solution of only the compound libraryin the binding buffer. Alternatively, the apparatus illustrated in FIG.4 can be used. As shown in FIG. 4, a plurality of reservoirs 27 (e.g.,syringes) are held in place with clamp 38. Each reservoir 27 contains amixture of a compound library and an indicator compound in a suitablediluent (or, alternatively, simply the indicator). The end of eachreservoir 27 is connected via tubing 29 to the inflow end of a column 30containing the target receptor bound to a solid phase support. Theoutflow end of each column 30 is connected via tubing 31 to anelectronically-actuated multiport stream selection valve 32 whichcontrols the flow of the effluent from columns 30. Using valve 32, theeffluent from the columns may be directed into a waste container 33, viatubing 34, or into mixing tee 35, via tubing 36. Mixing tee 35 is alsoconnected to reservoir 36, containing a supplemental diluent, via tubing37. The effluent from each column 30 is mixed with the supplementaldiluent from reservoir 36 in mixing tee 35 and the outflow is directedvia tubing 38 into an electrospray mass spectrometer 39. To control theflow from the reservoirs 27 into columns 30, a stand-off block 40 may beemployed. When pressure is applied to stand-off block 40 via, forexample, a pump, the plunger 41 of each reservoir 27 is individuallydepressed in sequence thereby infusing the contents of the reservoirthrough tubing 29 into the corresponding column 30. The effluentemerging from each column 30 is sequentially directed into massspectrometer 39 for analysis.

The apparatus of this invention also permit the absolute affinity ordissociation constant, K_(d), for certain individual members of acompound library to be readily determined. In this regard, ligandshaving an affinity for the target receptor break through the column atvolumes (i.e., break through times) related to their concentrations andK_(d) values, according to the following equation:${V_{x} - V_{0}} = \frac{B_{t}}{\lbrack X\rbrack_{0} + \left( K_{d} \right)_{x}}$

where B_(t) represents the dynamic binding capacity of the column; [X]₀is the infusion concentration of the ligand in the compound library;K_(d) is the dissociation constant for the ligand; V₀ is the voidvolume; and V_(x) represents the volume at the mid-point of the frontcorresponding to the break through of the ligand. This simple equationindicates that, once B_(t) and the concentration of the ligand areknown, the dissociation constant of a ligand can be determined from asingle measurement of its V_(x)-V₀. This equation strictly applies onlyin the case of a single ligand. In many cases, however, this equation ora modification of it can be applied to multiple ligands as well.

In order to determine B_(t), a representative compound, e.g., compoundX, is infused through the column at various concentrations and thecorresponding V_(x)-V₀ values measured. A plot of ([X](V-V₀))⁻¹ versus[X]⁻¹ is generated, where the y-intercept indicates the dynamic bindingcapacity of the column (B_(t)) (analogous to a Lineweaver-Burk plot).

Once the dynamic binding capacity of the column has been determined, thedissociation constants for individual members of the compound librarycan be determined from a single FC-MS run. For example, the K_(d) forcompounds where [X]<<(K_(d))_(x) is determined simply from B_(t)/(V-V₀).For those members of the library with a low dissociation constant,knowledge of their concentration or infusion of the compound library athigher dilution is required to determine K_(d).

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of this invention.Unless otherwise stated, all temperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

B_(t) = dynamic binding capacity ° C. = degrees Celsius cm = centimetereq. = equivalents FAB = fast atom bombardment FC = frontalchromatography g = grams K_(d) = dissociation constant L = liter MALDI =matrix-assisted laser desorption/ionization meq. = milliequivalent mg =milligram mL = milliliter mM = millimolar mmol = millimole MS = massspectrometry m/z = mass charge ratio N = normal PBS = phosphate bufferedsaline PEEK = poly(ether ether ketone) pmol = picomole TIC = total ionchromatogram μg = micrograms μL = microliter μm = micrometer μM =micromolar V₀ = void volume

Example 1 Screening of an Oligosaccharide Library Using FC-MS

In this example, a compound library containing a mixture of sixoligosaccharides was screened using frontal chromatography incombination with an electrospray mass spectrometer to determine therelative affinity of the oligosaccharides for a monoclonal antibody thatrecognizes the 3,6-dideoxy-D-galactose (abequose) epitope in Salmonellaparatyphi B O-antigens.

The compound library consisted of the following six oligosaccharides:αGalNAc(1→3)βGal-OGr (compound 1); αGal(1→3)[αFuc(1→2)]βGal-OGr(compound 2); αMan(1→3)[αMan(1→6)]βMan-OGr (compound 3);αAbe(1→3)αTal-OCH₃ (compound 4); αGal(1→2)[αAbe(1→3)]αMan-OCH₃ (compound5); andαGlc(1→4)βGlc(1→4)αGal(1→2)-[αAbe(1→3)]αMan(1→3)αGlc(1→4)βGlc-OCH₃(compound 6), wherein Gr═O(CH₂)₈CO₂CH₃. Compound 1-3 were obtained usingthe procedures described in U.S. Pat. No. 4,362,720 to R. U. Lemieux etal., issued Dec. 7, 1987; U.S. Pat. No. 4,137,401 to R. U. Lemieux etal, issued Jan. 30, 1979; and K. J. Kaur et al., “Use ofN-Acetylglucosaminyltransferases I and II in the Preparative Synthesisof Oligosaccharides”, Carbohydr. Res. 1991, 210, 145-153; respectively,the disclosures of which are incorporated herein by reference in theirentirety. Compounds 4-6 were obtained using the procedures described inD. R. Bundle et al., “Modulation of Antibody Affinity by SyntheticModifications of the Most Exposed Pyranose Residue of A TrisaccharideEpitope”, Bioorg. Med. Chem. 1994, 2, 1221-1229, the disclosure of whichis incorporated herein by reference in its entirety. Compounds 1-3 areknown to have no specificity for the antibody. On the other hand,compounds 4-6 contain the minimal requirement for recognition (abequose)and span a range of affinity for the antibody. The K_(d) values forcompounds 4-6, as determined by titration microcalorimetry, are shown inTable 1 below.

The monoclonal antibody used in this experiment was produced asdescribed in D. R. Bundle et al, “Molecular Recognition of a SalmonellaTrisaccharide Epitope by Monoclonal Antibody Se155.4” Biochem. 1994, 33,5172-5182. The antibody (0.5 mg) was biotinylated with a biotin reagentcontaining a long-chain spacer arm (NHS-LC-biotin, Pierce). The extentof biotin incorporation was monitored by matrix-assisted laserdesorption/ionization and the reaction was terminated at 14 biotins/IgG(average). The biotinylated antibody was then coupled to a beadedsupport by incubating the antibody with 25 μL of Ultralink immobilizedavidin (Pierce, Cat. No. 53119) in bicarbonate buffer (pH 8.5) for 1hour. The beads were then thoroughly washed with the bicarbonate buffer.A UV quantitation indicated an immobilization of ˜45 μg antibody/25 μLbeads was achieved. The beads were then slurry-packed into a 500 μm i.d.by 11.5 cm poly(ether ether ketone) (PEEK) column body (˜23 μL columnvolume).

In this experiment, a mixing tee served a dual role as a columnend-fitting and mixing chamber for the column eluent and organic make-upflow. The column was then directly connected to an electrospray massspectrometer (Hewlett-Packard series 1100 MSD, single quadrupole).

For operation in frontal chromatography mode, the column was firstflushed with ammonium acetate buffer (NH₄OAc, 2 mM, pH 6.7). Afterflushing , the flow was switched to a second solution containing amixture of the six oligosaccharides in ammonium acetate buffer, eachpresent at 1 μM. All solutions were infused concurrently with amulti-syringe pump (PHD 200, Harvard Apparatus) at a flow rate of 8μL/min/syringe (1 cc syringes). A Rheodyne valve (Model 9725) was usedfor flow switching. The column effluent combined with the make-up flow(10% 2 mM NH₄OAc buffer in acetonitrile) in the tee to provide a flowrate of 16 μL/min into the mass spectrometer.

For the analysis of this mixture, the spectrometer was scanned from m/z100-1500. Data was collected in scan mode with positive ion detection. Atotal ion chromatogram (TIC) was constructed from a 50 minute run timeas shown in FIG. 5A. This represented the consumption of only 400 pmolof each oligosaccharide. Peaks at specific m/z values were thenidentified through the analysis of the mass spectra giving rise to theTIC and selected ion chromatograms for all six compounds werereconstructed from the TIC as shown in FIG. 5B. Compounds 1-3 breakthrough the column simultaneously as indicated by the solid line. Massspectra were then generated from time-slices of the TIC (at times I, IIand III) as shown in FIGS. 5C, 5D and 5E. These mass spectra chart theprogression of the various oligosaccharides through the column. Aspectrum representing the onset of compound 4 is not shown.

As discussed above, ligands having no affinity for the target receptorbreak through at the void volume (V₀), while compounds having anaffinity for the target ligand break through later, at volumes relatingto their concentrations and K_(d) values, according to the followingequation:${V_{x} - V_{0}} = \frac{B_{t}}{\lbrack X\rbrack_{0} + \left( K_{d} \right)_{x}}$

where B_(t) represents the dynamic binding capacity of the column; [X]₀is the infusion concentration of the ligand in the compound library;K_(d) is the dissociation constant for the ligand; V₀ is the voidvolume; and V_(x) represents the volume at the mid-point of the frontcorresponding to the break through of the ligand.

In order to determine B_(t), compound 5 was infused through the columnat various concentrations and the corresponding V-V₀ values measured. Aplot of ([A]_(O)(V-V₀))⁻¹ versus [A]₀ ⁻¹ was generated, where A iscompound 5, as shown in FIG. 6. The y-intercept indicated a B_(t) of 520pmol. Each antibody molecule contains two binding sites, therefore thiscorresponds to an active capacity of 260 pmol of protein (representing93% of the total amount of protein bound). The x-intercept indicated aK_(d) of 11.2 μM for compound 5, which compares favorably with the valuedetermined by microcalorimetry as shown in Table 1.

Knowledge of the column capacity prior to the screening of a mixtureallows for the determination of dissociation constants from a singlefrontal chromatogram. For compounds with [X]<<(K_(d))_(x), the K_(d) canbe determined simply from B_(t)/(V-V₀). For example, compound 4 wasshown to have a K_(d) of 0.2 mM, as determined from the chromatogram ofFIG. 5B. Compounds with low dissociation constants require either theknowledge of their concentration or the infusion of the mixture athigher dilution for the determination of K_(d). The K_(d) of compound 6,at a 1 μM concentration, was determined from the same chromatogram to be1.5 μM.

The column was regenerated offline by washing with a large volume ofbinding buffer. The column used in this example was subjected to over150 runs with no observable loss of activity or leaching of theantibody.

The results from this experiment are shown in Table 1.

TABLE 1 K_(d) ± s (μM)² Oligosaccharide Micro- FC/MS No. Gr =O(CH₂)₈CO₂CH₃ (MNa)⁺¹ cal³ Ind.⁴ Mix⁵ 1 αGalNAc(1 → 3)βGal-OGr 576.3 — —2 αGal(1 → 3)[αFuc(1 → 2)]βGal-Gr 681.3 — — 3 αMan(1 → 3)[αMan(1 →6)]βMan-OGr 697.3 — — 4 αAbe(1 → 3)αTal-OCH₃ 347.0 190 185 ± 17  178 ±23  5 αGal(1 → 2)[αAbe(1 → 3)]αMan-OCH₃ 509.2 6.3 12.6 ± 1.3  10.2 ±1.1  6 αGlc(1 → 4)βGlc(1 → 4)αGal(1 → 2)[αAbe 1157.4 0.88 1.79 ± 0.201.71 ± 0.16 (1 → 3)αMan(1 → 3)αGlc(1 → 4)βGlc- OCH₃ ¹Monoisotopicmolecular weight of the singly charged sodium adduct. ²Dissociationconstant with the corresponding standard deviation. ³Microcalorimetry.⁴Values determined from infusion of individual ligand, with Compounds1-3. ⁵Values determined from the infusion of the six-compound mixture.

The results in Table 1 demonstrate that the affinity of various putativeligands in a compound library for a target receptor can be determinedrelative to other putative ligands in the library; and that thedissociation constant, K_(d), for putative ligands and the targetreceptor can be determined. The results further demonstrate that thereis an acceptable correlation between the literature K_(d) values andthose generated by FC-MS procedures.

Example 2 Screening of an Oligosaccharide Library Using FC-MS and anIndicator Compound

In this example, the use of an indicator compound to screen a compoundlibrary is demonstrated. The antibody used in this example was the sameas that used in Example 1, i.e., a monoclonal antibody that recognizesthe 3,6-dideoxy-D-galactose (abequose) epitope in Salmonella paratyphi BO-antigens. The column was also essentially the same as the column inExample 1 and it was prepared and operated as described therein.

In this experiment, three solutions were prepared. Solution A containedthe following four oligosaccharide in 2 mM NH₄OAc: αGalNAc(1→3)βGal-OGr(compound 1); αGal(1→3)[αFuc(1→2)]βGal-OGr (compound 2);αMan(1→3)[αMan(1→6)]βMan-OGr (compound 3); αAbe(1→3)αTal-OCH₃ (compound4), wherein Gr═O(CH₂)₈COCH₃. Solution B containedαGal(1→2)[αAbe(1→3)]αMan-OCH₃ (compound 5) in 2 mM NH₄OAc; and SolutionC contained compounds 1-5 in 2 mM NH₄OAc. In all solutions, compounds 1,2 and 3 were present at 1 μM, compound 4 was present at 0.16 μM, andcompound 5 was present at 15 μM. In this example, compound 4 was used asthe indicator compound and compound 5 was used to represented a memberof a compound library. The remaining compounds were used to determineV_(o).

Solution A containing compounds 1-4 was infused into the column asdescribed in Example 1. A quadrupole mass spectrometer was used tomonitor the effluent. The mass spectrometer was operated in selected ionmonitoring (SIM) mode, on the (M+Na)⁺ peak of each compound. FIG. 5Ashows the selected ion chromatograms generated from an infusion ofcompounds 1-4 (i.e., Solution A). The breakthrough volume for compound 4was 3.0±0.1 μL. The column was regenerated by flushing with the bindingbuffer (i.e., 2 mM NH₄OAc) for about 10 min. at which time essentiallyall traces of compound 4 were removed.

Using the apparatus of FIG. 1, Solution B (compound 5) and Solution C(compounds 1-5) were loaded into separate syringes. Solution B wasinfused through the column until dynamic equilibrium for compound 5 wasattained. At this point, the flow was switched to the syringe carryingSolution C, and the selected ion chromatograms of FIG. 7B were generatedusing the quadrupole mass spectrometer. As shown in FIG. 7B,pre-equilibration of the column with compound 5 leads to a measurableshift in the breakthrough volume of the indicator compound 4 (to 1.1±0.3μl). This is consistent with the fact that compound 5 is a ligand havinga K_(d) for the antibody lower than that of the indicator compound 4(see Table 1 above). Therefore, by simply monitoring the indicatorcompound, the fact that the representative library contained a compoundwith a higher affinity for the target receptor was readily apparent.

Note that while the indicator compound (compound 4) in this experimentwas added to a solution of the representative library (compound 5), thiswill not always be necessary. In those situations where the library(Solution B) contains a strongly retained compound (i.e., low K_(d), oroff-rate), Solution A can be substituted for Solution C (i.e., theindicator does not need to be mixed with the library).

Example 3 Screening of an Oligosaccharide Library Using FC-MS

In this example, a compound library containing a mixture of fouroligosaccharides was screened using frontal chromatography incombination with an electrospray mass spectrometer to determine therelative affinity of the oligosaccharides for cholera toxin B subunit.

The compound library consisted of the following four oligosaccharides:αGalNAc(1→3)βGal-OGr (compound 1); αGal(1→3)[αFuc(1→2)]βGal-OGr(compound 2); αMan(1→3)[αMan(1→6)]βMan-OGr (compound 3); and GM₁oligosaccharide (compound 7, wherein Gr═O(CH₂)₈CO₂CH₃. Compound 7, whichis the natural ligand for cholera toxin B subunit, was obtained usingthe procedures described in A. Schön et al., “Thermodynamics ofIntersubunit Interactions in Cholera Toxin upon Binding to theOligosaccharide Portion of Its Cell Surface Receptor, GangliosideG_(M1)” Biochem. 1989, 28, 5019-5024, the disclosure of which isincorporated herein by reference in its entirety. Cholera toxin Bsubunit was obtained from LIST Biochemicals, Campbell, Calif.

A column was prepared from a 12 cm section of 0.01″ (250 μm) i.d. PEEKtubing (column volume of about 6 μL). The column was packed with POROS20 immobilized streptavidin particles (available from PerseptiveBiosystems, Framingham, Mass.).

Cholera toxin B subunit (a pentameric protein) was biotinylated toprovide about 1-2 biotins/monomer, as measured by MALDI. A dilutesolution of this biotinylated protein (4 μM) was infused through thepre-packed column such that the total amount of cholera toxin B subunitbound was approximately 200 pmol after washing (as determined by UVquantitation).

A solution containing compounds 1-3 and 7 was prepared. All compoundswere present at 2 μM, in 2 mM NH₄OAc (pH 6.9). Using an apparatussimilar to that shown in FIG. 1, the column was first equilibrated withthe binding buffer (2 mM NH₄OAc). The solution containing compounds 1-3and 7 was then infused through the column at 8 μL/min. The effluent wascombined with a typical make-up flow (10% 2mM NH₄OAc in acetonitrile)and passed into an electrospray single quadrupole mass spectrometer.Data was collected in scan mode, with negative ion detection.

A total ion chromatogram was generated, followed by reconstruction ofselected ion chromatograms for each of compounds 1-3 and 7 as shown inFIG. 8. As illustrated in FIG. 8, compounds 1-3 broke through in thevoid volume of the system (˜4 min×8 μL/min=32 μL) while compound 7 (GM₁oligosaccharide) broke through at ˜300 μL. Thus, GM₁ oligosaccharide(K_(d)≅100 nM) has a stronger affinity for cholera toxin B subunit thancompounds 1-3 which have little or no affinity for cholera toxin Bsubunit.

A second mixture was then prepared in the binding buffer and analyzed byFC-MS in a similar fashion. This mixture contained a syntheticallyprepared GM₁ analogue, i.e., βGal(1→3)βGalNAc(1→)-OCH₂CH₂O-(←2)αNeu5Ac,(compound 8) in an impure form (i.e. containing unidentifiedintermediates and reaction byproducts). Compound 8 was prepared by themethods described in P. Füigedi et al, “A Novel Promoter for theEfficient Construction of 1,2-trans Linkages in Glycoside Synthesis,Using Thioglycosides as Glycosyl Donors” Carbohydr. Res. 1986, 149,C9-C12; A. Marra et al., Stereoselective Synthesis of 2-Thioglycosidesof N-Acetylneuraminic Acid“, Carbohydr. Res. 1989, 187, 35-42; and L.Lay et al., “Synthesis of the Propyl Glycoside of the Trisaccharideα-L-Fucp-(1→2)-β-D-Galp-(1→3)-β-D-GalpNAc. Components of a Tumor AntigenRecognized by the Antibody Mbr1” Helv. Chim. Acta. 1994, 77, 509-514;the disclosures of which are incorporated herein by reference in theirentirety. The mixture was infused through the column, and the massspectrometer was set to operate in selected ion monitoring mode, onnegative ions representative of compounds 3 and 8. Selected ionchromatograms were generated for these ions as shown in FIG. 9. FIG. 9shows that compound 3 broke through in the void volume (m/z 673.2). Amore complex pattern was observed for the ions with a mass/charge of717.2 u. A certain fraction of these ions also broke through in the voidvolume (˜25%), while the remaining 75% broke through significantly later(at about 11 min). This two-front profile indicates an isobaric impurityexists at the 25% level, which does not bind to cholera toxin B subunit.Thus, FC-MS is able to ascertain the presence of isobaric, non-bindingimpurities. Reasonably accurate quantitation of these impurities canalso be achieved.

Example 4 Screening of a Compound Library Containing 100 PutativeLigands Against a Human Enzyme

In this example, a compound library containing a mixture of 100tripeptides was screened against immobilized human α-thrombin usingfrontal chromatography in combination with an electrospray massspectrometer. The peptides were synthesized as a mixture by establishedsolid phase techniques and were purchased from Alberta Peptide Institute(Edmonton, Alberta, Canada). This set of peptides all have a commonC-terminal amino acid (arginine), while the remaining two positions arerandom and chosen from a set of 10 amino acids (see FIG. 12). FIG. 12also displays an electrospray mass spectrum of the mixture. The spectrumwas collected from an infusion of a 50 μM solution in 1:1acetonitrile:ammonium acetate (2 mM, pH 7.2). Assuming equimolar ratiosof all peptides, this corresponds to 0.5 μM per peptide. This spectrumhighlights a peak at m/z of 419.2. This peak corresponds to two isomericentries in the library: PfR and fPR, where f refers to D-phenylalanine.The tripeptide fPR has been identified in the literature as an inhibitorpossessing a K_(d) value of approximately 1 μM against human α-thrombin.An experiment was conducted to determine if this ligand could bedetected when present in the full mixture as screened against a thrombincolumn.

A column was prepared from a 5 cm section of 0.01″ (250 μm i.d.) PEEKtubing (column volume of about 2.5 μL). The column was packed with POROS20 immobilized streptavidin particles (available from PerseptiveBiosystems, Framingham, Mass.). Human α-thrombin was purchased fromSigma Chemical Co., and biotinylated with a reagent containing along-chain spacer arm (sulfo-NHS-LC-biotin, Pierce). The extent ofbiotin incorporation was less than 5 biotins/thrombin, as monitored bymatrix-assisted laser desorption/ionization. A dilute solution of thisbiotinylated protein (approximately 2 μM) was infused through thepre-packed column in the presence of 0.1% bovine serum albumin (w/v)such that the total amount of immobilized thrombin was approximately 570pmol (as calculated by a determination of the reduced capacity of thecolumn for free biotin).

The BOC-protected fPR (BOC-fPR, also known as ligand) was purchased fromCalbiochem and infused through the column at a concentration of 1 μM inammonium acetate solution (2 mM, pH 7.2), and at a flow rate of 8μL/min. FIG. 13 displays a chromatogram reflecting three infusions/washcycles of this peptide through the column, in the presence of a voidmarker compound (a non-binding trimannosyl compound). FIG. 13 shows theselected ion chromatograms for each compound. The average V-V₀ value wasdetermined to be 4.66 μL. Based on this experiment, and the infusion ofthis peptide at higher concentrations, the B_(t) value of the column wascalculated to be approximately 145 pmol (˜25% active). The BOC-fPR wasselected as the indicator compound for the following work, with thetrimannosyl compound the corresponding void marker compound.

The library of peptides was then infused through the column at aconcentration of 1 μM per peptide for approximately 30 minutes,whereupon the indicator compound and void market compound (in thepresence of the library) were infused. The same buffer and flow rateconditions as above were used. FIG. 14 displays the V-V₀ valueimmediately before (14A) and immediately after (14B) the 30 minuteequilibration time. The drop in V-V₀ as a result of the 100 peptidesindicates the loss of virtually all of the binding activity of theprotein.

To determine the nature of the compounds giving rise to the indicatorshift, the peptide library was infused through a fresh, identicallyprepared column in FC/MS mode. The HP quadrupole electrospray massspectrometer was set to scan the mass range from m/z 100 to 600 at ascan rate of approximately 1 sec/cycle. The effluent was monitored inreal time, and the experiment was stopped at approximately 15 minutes.The results are displayed in FIGS. 15A and 15B. FIG. 15A shows afeatureless total ion chromatogram. However, a generation of selectedion chromatograms from the peaks representing the mixture componentsresulted in the identification of m/z 419.2 as giving rise to thelargest V-V₀ shift (as shown in FIG. 15B). Two breakthrough curves areevident in this Figure, indicating the presence of at least two isomers.Based on a knowledge of the mixture composition, this is consistent withthe presence of the isomers PfR (non-binding) and fPR (the ligand). Afresh column was constructed through which a solution of just the voidmarker compound and fPR (1 μM each) was infused. This generated a V-V₀that was approximately twice the value measured from the mixture. Asimilar experiment was conducted using PfR instead, which generated nomeasurable V-V₀. This confirms that fPR is indeed the ligand in themixture. The combination of a large indicator shift and a smaller thanexpected V-V₀ for the ligand is consistent with the presence ofadditional ligands. Thrombin is a serine protease capable of cleavingthe C-terminal side of K and R, therefore a large fraction of thesepeptides serve as substrates for the enzyme. At the infusionconcentrations of the experiment, these peptides compete with thebinding of fPR. This shows that FC/MS, in conjunction with the indicatoranalysis, serve as a rapid means of identifying ligands from mixtures.

An additional experiment was conducted with this system to illustratethe use of the roll-up effect in determining the presence of a strongligand. A thrombin column similar in construct to those mentioned abovewas prepared, this time containing approximately 50 pmol of activeprotein. The ligand fPR was selected as an indicator and infused throughthe column at a concentration of 1 μM to generate the selected ionchromatogram of FIG. 16A. This represents a typical break through curve.The column was regenerated offline with binding buffer, whereupon asolution containing 1 μM of fPR and 1 μM of fPR-chloromethyl ketone (anaffinity label with a K_(d) value of approximately 50 nM) was infused.The selected ion chromatogram is shown in FIG. 16B, where the solid linerepresents fPR and the dashed line fPR-chloromethyl ketone. For clarity,the selected ion chromatogram for the void marker is not displayed.Firstly, there is a time shift in the break through curve for fPR vs.the break through curve of FIG. 16A. Secondly, the y axis of FIG. 16Bindicates a maximum intensity of approximately 3 times that of theinfusion concentration, followed by a return to the infusionconcentration level. This indicates the presence of a stronger ligand inthe mixture that causes the release of prebound indicator (thisindicator loaded on to the column ahead of the stronger ligand). Notethat the peak correlates with the onset of the break through time forthe stronger ligand (dashed line). Therefore, monitoring solely theindicator leads to the identification of a mixture containing at leaston ligand with a binding constant lower than the indicator.

From the foregoing description, various modifications and changes in thecomposition and method will occur to those skilled in the art. All suchmodifications coming within the scope of the appended claims areintended to be included therein.

What is claimed is:
 1. An apparatus for screening a plurality of targetreceptors to determine the relative affinity of the receptors to animmobilized ligand or ligands relative to an indicator compound or aplurality of indicator compounds, which apparatus comprises: (a) aplurality of columns each column comprising a ligand or a plurality ofligands wherein each ligand is bound to a solid phase support, and eachcolumn having a inflow end and an outflow end, wherein each of saidcolumns is capable of independently having a target receptor or aplurality of target receptors applied thereto under frontalchromatography conditions to produce an effluent from the outflow end ofthe column; (b) a plurality of first reservoirs each connected to theinflow end of one of the columns for applying a target receptor or aplurality of target receptors to the columns; (c) a plurality of secondreservoirs each connected to the inflow end of one of the columns forapplying either (i) a mixture comprising the target receptor orplurality of target receptors, at least one void marker compound and anindicator compound or a plurality of indicator compounds, (ii) at leastone void marker compound and an indicator compound or a plurality ofindicator compounds, or (iii) a buffer solution to the column; (d) amass spectrometer connected to the outflow end of each of said columnsfor intermittently analyzing the effluent from each of the column. 2.The apparatus of claim 1, wherein said apparatus further comprises: (e)a third reservoir connected to the outflow end of each of the columnsfor supplying a supplemental diluent to the effluent from each columnbefore analysis by the mass spectrometer.
 3. The apparatus of claim 1wherein said apparatus comprises from 2 to about 100 columns.
 4. Theapparatus of claim 3, wherein said apparatus comprises from 3 to about50 columns.
 5. The apparatus of claim 4, wherein said apparatuscomprises from 5 to about 10 columns.
 6. The apparatus of claim 1,wherein each column is intermittently monitored for a period of about0.5 seconds to about 10 seconds before switching to the next column. 7.The apparatus of claim 6, wherein each column is intermittentlymonitored for about 1 second to about 5 seconds before switching to thenext column.
 8. The apparatus of claim 1, wherein the column has aninternal diameter ranging from about 10 μm to about 4.6 mm.
 9. Theapparatus of claim 8, wherein the column has an internal diameter offrom about 100 μm to about 250 μm.
 10. The apparatus of claim 1, whereinthe column has a length of from about 1 cm to about 30 cm.
 11. Theapparatus of claim 1, wherein the column has a length of from about 2 cmto about 20 cm.
 12. The apparatus of claim 1, wherein each ligand isselected from the group consisting of carbohydrates, monosaccharides,oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides,polypeptides, proteins, nucleosides, nucleotides, oligonucleotides,polynucleotides, lipids, retinoids, steroids, glycopeptides,glycoproteins, glycolipids, proteoglycans, and synthetic analogs orderivatives thereof.
 13. The apparatus of claim 1, wherein each ligandis selected from the group consisting of synthetic small moleculeorganic compounds.